Quality control filtering of the data was performed, and two samples from each of the phosphorylation and protein abundance datasets were removed because of poor correlation with their respective replicates (Figures S1 A and S1B). Principal component analysis (PCA) of the remaining samples revealed good separation of mock and infected samples as well as high quantitative reproducibility between biological replicates (Figures 1B, 1C, and S1C). In total, high-quality quantification of 4,624 human-orthologous phosphorylation sites and 3,036 human-orthologous proteins was obtained (Figure S1D). Successful infection was confirmed by the observation of a dramatic increase in viral protein abundance over the course of a 24-h infection period (Figures 1D and S1E).
Figure S1 Proteomics: Quality Control (QC), Orthology, Enrichments, and Viral Proteins, Related to Figure 1
(A) Principal component analysis computed on intensities summarized by MSstats at the level of phosphorylation site groups within (from left to right) all runs, with one outlier run removed, and with two outlier runs removed. Outlier runs are labeled 00Hr.2 and 02.Hr.2. (B) Principal components analysis computed on protein intensities as summarized by MSstats (from left to right) within all runs, with one outlier run removed, and with two outlier runs removed. Outlier runs are both labeled 00Hr.2; one is mock and the other is infected. (C) Coefficient of variance boxplot for each condition. Black lines depict the median and their values are indicated above each boxplot. (D) Mapping detected and quantifiable proteins and phosphorylation sites from the green monkey (Chlorocebus sabaeus) protein sequences to human genes. Proteins and sites were considered quantifiable if MSstats computed a non-infinite fold change for any time point or if an infinite log2 fold change passes criteria for inclusion in any time point. (E) Intensities of viral proteins as summarized over all peptide ion fragments by MSstats, averaged across replicates. The MSstats summarization is based on the median intensity of all fragments after data pre-processing (STAR Methods). (F) Gene Ontology enrichment analysis for proteins significantly regulated in terms of abundance upon infection, separated by time point and direction of phosphorylation regulation. All terms with significant over-representation (adjusted p value < 0.01) in the regulated gene set are kept, and redundant terms are removed (see STAR Methods). Numbers in cells indicate the number of genes that match the term for a given time point and direction. (G) Gene Ontology enrichment analysis for significantly phosphorylated proteins upon infection, separated by time point and direction of protein regulation. Details same as for (F).